Guarding the Genome

The active site of the tyrosine family site-specific recombinase Flp contains a conserved catalytic pentad that includes two arginine residues, Arg-191 and Arg-308. Both arginines are essential for the transesterification steps of strand cleavage and strand joining in DNA substrates containing a phosphate group at the scissile position. During strand cleavage, the active site tyrosine supplies the nucleophile to form a covalent 3-phosphotyrosyl intermediate. The 5-hydroxyl group produced by cleavage provides the nucleophile to re-form a 3-5 phosphodiester bond in a recombinant DNA strand. In previous work we showed that substitution of the scissile phosphate (P) by the charge neutral methylphosphonate (MeP) makes Arg-308 dispensable during the catalytic activation of the MeP diester bond. However, in the Flp(R308A) reaction, water out-competes the tyrosine nucleophile (Tyr-343) to cause direct hydrolysis of the MeP diester bond. We now report that for MeP activation Arg-191 is also not required. In contrast to Flp(R308A), Flp(R191A) primarily mediates normal cleavage by Tyr-343 but also exhibits a weaker direct hydrolytic activity. The cleaved MeP-tyrosyl intermediate formed by Flp(R191A) can be targeted for nucleophilic attack by a 5-hydroxyl or water and channeled toward strand joining or hydrolysis, respectively. In collaboration with wild type Flp, Flp(R191A) promotes strand exchange between MeP-and P-DNA partners. Loss of a catalytically crucial positively charged side chain can thus be suppressed by a compensatory modification in the DNA substrate that neutralizes the negative charge on the scissile phosphate.The hallmark of the active sites of tyrosine site-specific recombinases and type IB topoisomerases is a catalytic pentad (Arg-Lys-(His/Lys)-Arg-(His/Trp)), with a tyrosine residue providing the nucleophile for DNA cleavage (1-6). The primary function of the conserved arginines Arg-191 and Arg-308 in Flp, inferred from crystallographic data, appears to be in balancing the phosphate negative charge in the transition state during the strand cleavage and strand joining steps of DNA recombination and relaxation, respectively. Replacement of alanine, glycine, or the conservative lysine at either of these positions results in the gross impairment of the catalytic activities of the corresponding Flp variants (7-10). Recent studies show that substitution of the scissile phosphate (P) 3 by methylphosphonate (MeP), which is charge neutral in the ground state and carries a nominal Ϫ1 charge in the transition state, makes Arg-308 dispensable in the MeP activation step by Flp (11). The same holds for the corresponding arginine residues of the Cre recombinase (Arg-292) and vaccinia topoisomerase (Arg-223) (12, 13). The chemical competence of Cre(R292A), Flp(R308A), and Topo(R223A) on their respective MeP substrates is consistent with the suspected electrophilic role of the positively charged arginine side chain in catalysis.Cre(R292A), wild type topoisomerase, and Topo(R223A) cleave the MeP diester bond using their ...

Section: Hydrolysis Of Mepmentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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Electrostatic Suppression Allows Tyrosine Site-specific Recombination in the Absence of a Conserved Catalytic Arginine

Rowley

Kachroo

Hui

et al. 2010

“…(The Tp2 nucleotide is defined as the ϩ1 nucleotide.) Four conserved amino acids (Arg-130, Lys-167, Arg-223, and His-265) are responsible for catalyzing the attack of the active site tyrosine (Tyr-274) on the scissile phosphodiester to form the covalent intermediate (2)(3)(4)(5)(6). Biochemical and structural studies suggest that recognition of the 5Ј-CCCTT/3Ј-GGGAA sequence triggers conformational changes in the enzyme that recruit the full set of catalytic side chains into the active site, a process that entails protein contacts with several of the nucleotide bases and specific atoms of the phosphate backbone of DNA within and immediately flanking the CCCTT element (7)(8)(9)(10)(11)(12).…”

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confidence: 99%

Nonpolar Nucleobase Analogs Illuminate Requirements for Site-specific DNA Cleavage by Vaccinia Topoisomerase

Yakovleva

Lai

Kool

et al. 2006

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Vaccinia DNA topoisomerase forms a covalent DNA-(3-phosphotyrosyl)-enzyme intermediate at a specific target site 5-C ؉5 C ؉4 C ؉3 T ؉2 T ؉1 p2N ؊1 in duplex DNA. Here we study the effects of nonpolar pyrimidine isosteres difluorotoluene (F) and monofluorotoluene (D) and the nonpolar purine analog indole at individual positions of the scissile and nonscissile strands on the rate of single-turnover DNA transesterification and the cleavage-religation equilibrium. Comparison of the effects of nonpolar base substitution to the effects of abasic lesions reported previously allowed us to surmise the relative contributions of base-stacking and polar edge interactions to the DNA transesterification reactions. For example, the deleterious effects of eliminating the ؉2T base on the scissile strand were rectified by introducing the nonpolar F isostere, whereas the requirement for the ؉1T base was not elided by F substitution. We impute a role for ؉1T in recruiting the catalytic residue Lys-167 to the active site. Topoisomerase is especially sensitive to suppression of DNA cleavage upon elimination of the ؉4G and ؉3G bases of the nonscissile strand. Indole provided little or no gain of function relative to abasic lesions. Inosine substitutions for ؉4G and ؉3G had no effect on transesterification rate, implying that the guanine exocyclic amine is not a critical determinant of DNA cleavage. Prior studies of 2-aminopurine and 7-deazaguanine effects had shown that the O6 and N7 of guanine were also not critical. These findings suggest that either the topoisomerase makes functionally redundant contacts with polar atoms (likely via Tyr-136, a residue important for precleavage active site assembly) or that it relies on contacts to N1 or N3 of the purine ring. The cleavage-religation equilibrium is strongly skewed toward trapping of the covalent intermediate by elimination of the ؉1A base of the nonscissile strand; the reaction equilibrium is restored by ؉1 indole, signifying that base stacking flanking the nick is critical for the religation step. Our findings highlight base isosteres as valuable tools for the analysis of proteins that act on DNA in a site-specific manner.Vaccinia virus DNA topoisomerase IB cleaves and rejoins one strand of the DNA duplex through a transient DNA-(3Ј-phosphotyrosyl)-enzyme intermediate formed at a specific pentapyrimidine target sequence, 5Ј-(T/C)CCTTp2 (1). (The Tp2 nucleotide is defined as the ϩ1 nucleotide.) Four conserved amino acids (Arg-130, Lys-167, Arg-223, and His-265) are responsible for catalyzing the attack of the active site tyrosine (Tyr-274) on the scissile phosphodiester to form the covalent intermediate (2-6). Biochemical and structural studies suggest that recognition of the 5Ј-CCCTT/3Ј-GGGAA sequence triggers conformational changes in the enzyme that recruit the full set of catalytic side chains into the active site, a process that entails protein contacts with several of the nucleotide bases and specific atoms of the phosphate backbone of DNA within and immediately flanking the C...

“…The two arginines and the histidine interact directly with the scissile phosphodiester ( Fig. 1) and are proposed to stabilize the developing negative charge on a putative pentacoordinate phosphorane transition state (11)(12)(13)(14)(15). Lys 167 serves together with Arg 220 as a general acid to expel the 5Ј-OH leaving group during the cleavage reaction (16,17).…”

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confidence: 99%

Chemical and Traditional Mutagenesis of Vaccinia DNA Topoisomerase Provides Insights to Cleavage Site Recognition and Transesterification Chemistry

Yakovleva

Chen

Hecht

et al. 2008

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Vaccinia DNA topoisomerase IB (TopIB) relaxes supercoils by forming and resealing a covalent DNA-(3-phosphotyrosyl)-enzyme intermediate. Here we gained new insights to the TopIB mechanism through "chemical mutagenesis." Meta-substituted analogs of Tyr 274 were introduced by in vitro translation in the presence of a chemically misacylated tRNA. We report that a meta-OH reduced the rate of DNA cleavage 130-fold without affecting the rate of religation. By contrast, meta-OCH 3 and NO 2 groups elicited only a 6-fold decrement in cleavage rate. We propose that the meta-OH uniquely suppresses deprotonation of the para-OH nucleophile during the cleavage step. Assembly of the vaccinia TopIB active site is triggered by protein contacts with a specific DNA sequence 5- interact with purine nucleobases in the major groove. Whereas none of these side chains is essential per se, an N140A/T142A double mutation reduces the rate of supercoil relaxation and DNA cleavage by 120-and 30-fold, respectively, and a K133A/K135A double mutation slows relaxation and cleavage by 120-and 35-fold, respectively. These results underscore functional redundancy at the TopIB-DNA interface.Type IB DNA topoisomerases (TopIB) 2 relax DNA supercoils by repeatedly breaking and rejoining one strand of the DNA duplex through a covalent DNA-(3Ј-phosphotyrosyl)-enzyme intermediate. Vaccinia virus TopIB, which displays stringent specificity for cleavage at the sequence 5Ј(C/T)CCTT2 in the scissile strand (1-5), has been an instructive model system for mechanistic studies of the TopIB family. Analyses of the effects of vaccinia TopIB mutations and DNA target site modifications revealed that the cleavage and religation transesterification reactions are driven by four conserved amino acid side chains (Arg 130 , Lys 167 , Arg 223 , and His 265 ) that compose the active site and catalyze the attack of Tyr 274 on the scissile phosphodiester (6 -10). The two arginines and the histidine interact directly with the scissile phosphodiester ( Fig. 1) and are proposed to stabilize the developing negative charge on a putative pentacoordinate phosphorane transition state (11-15). Lys 167 serves together with Arg 220 as a general acid to expel the 5Ј-OH leaving group during the cleavage reaction (16,17). One of the outstanding problems in TopIB biochemistry concerns whether and how the enzyme might activate the tyrosine nucleophile via general base catalysis.The poxvirus TopIB active site is not preassembled prior to DNA binding. The apoenzyme crystal structure of vaccinia TopIB showed that three of the catalytic residues (Arg 130 , Lys 167 , and Tyr 274) are either disordered or out of position to perform transesterification chemistry (18). Tyr 274 in the apoenzyme is located far away from the scissile phosphate and the other catalytic amino acids (Fig. 2A). The act of DNA binding elicits a major rearrangement of Tyr 274 and the surrounding ␣-helix that brings Tyr 274 into the active site in proximity to Arg 223 . The conformational switch occurs by melting a long ␣-heli...